Fig. 1. Cell-type diversity in the mouse hypothalamus.

(a) Workflow diagram to indiscriminately obtain intact single cells from the juvenile mouse hypothalamus and analyze them without selection. A central vertical column spanning the preoptic area and arcuate nucleus in its rostrocaudal extent (yellow shading) was dissected, enzymatically dissociated and processed for single-cell RNA-seq. Molecule counts from 3131 cells were first clustered to define main cell types. Heat-map shows blocks of genes enriched in each cell type. (b) mRNA transcripts specifying each of the 7 major cell types. Dot density plots depict means (bars) and individual values (circles). mRNA expression of selected known and novel markers. A marker pair is shown per class including a generally accepted and a lesser-known gene. Abbreviations: Astroc., astrocytes; Epend., ependymal cells; Microg., microglia; Olig., oligodendrocytes; Vsm, vascular and smooth muscle lineage; Endoth., endothelial cells. (c) Clustering of 898 neurons reveals unprecedented molecular diversity. Heat-map shows clustering results with blocks of clusters aligned along the diagonal axis and boxed in orange. Abbreviations in inset denote subtype-specific neuropeptides and hormones. (d) Visualization of hypothalamic neuron subtypes on a two-dimensional map using tSNE (1,194 genes, perplexity = 5, 200 principle components; see also Supplementary Figs. 3,4). Neurons were color-coded by highest expression of well-known, cluster-defining hypothalamic markers. (d₁) Pomc mRNA expression is an example of phenotypic clustering on the same tSNE plot. (d₂) In contrast, Crh is heterogeneously distributed on the tSNE scaffold, precluding its use to typify neurons. Abbreviations in (c,d): Agrp, agouti-related peptide; Avp, arginine-vasopressin; Bdnf, brain-derived neurotrophic factor; Gad1, glutamate decarboxylase 1; Gal, galanin; Ghrh, gonadotropin-releasing hormone; Hcrt, hypocretin; Nms, neuromedin S; Npvf, neuropeptide VF precursor; Npy, neuropeptide Y; Oxt, oxytocin; Pmch, pro-melanin-concentrating hormone; Pomc, pro-opiomelanocortin; Qrfp, RF(Arg-Phe)amide family 26 amino acid peptide; Slc17a6, vesicular glutamate transporter 2; Sst, somatostatin; Th, tyrosine hydroxylase; Trh, thyrotropin-releasing hormone; Vip, vasoactive intestinal polypeptide.